The appetite-stimulating peptide hormone, ghrelin, is the only protein in animals that is known to be modified by O-acylation with octanoate, an eight-carbon fatty acid. Octanoylation is required for the endocrine actions of ghrelin, but no enzyme that catalyzes this novel modification has yet been identified (Kojima and Kangawa, 2005; van der Lely et al., 2004).
The discovery of ghrelin was reported in 1999 by Kojima et al. (Kojima et al., 1999), who were searching for a ligand for an orphan G-protein coupled receptor (GHS-R) that stimulates the secretion of growth hormone in the pituitary gland. The ligand was purified from rat stomach, and it was shown to stimulate the release of growth hormone from cultured pituitary cells. Kojima, et al. (1999) determined that the 28-amino acid ghrelin is derived proteolytically from a precursor of 117 amino acids. Analysis by mass spectroscopy revealed that serine-3 of ghrelin is modified by O-acylation with an octanoyl residue, which is required for growth hormone releasing activity. Serine-3 is conserved in mammals, birds, and fish. In the bullfrog serine-3 is replaced by threonine, but this residue is also octanoylated (Kaiya et al., 2001; Kojima and Kangawa, 2005). Thus, O-octanoylation of ghrelin has been conserved in vertebrates over millions of years of evolution.
Interest in ghrelin rose dramatically when it was demonstrated that ghrelin concentrations in human plasma rise immediately before mealtimes (Cummings, 2006; Small and Bloom, 2004). Moreover, infusion of ghrelin into the cerebral ventricles of rats markedly enhances food intake apparently through actions on the hypothalamus (Kamegai et al., 2001). Elimination of ghrelin or its receptor in mice through knockout technology caused a modest but significant reduction in obesity when the mice were presented with high fat diets (Wortley et al., 2005; Zigman et al., 2005). These findings aroused interest in ghrelin inhibitors as potential preventatives for obesity in humans.
One way to inhibit the action of ghrelin would be to block the supposed enzyme that attaches octanoate. An inhibitor should be quite specific since no other protein is known to be octanoylated. Thus far, however, a ghrelin octanoylating enzyme has escaped identification. In the current studies, we have identified the ghrelin-acylating enzyme.
The initial insight came from studies on the Drosophila wingless gene and its mammalian homolog, Wnt. Genetic studies in Drosophila had earlier demonstrated that Wingless activity required the action of another gene porcupine (Kadowaki et al., 1996). The amino acid sequence of Porcupine contains a conserved region that is found in a family of membrane-bound hydrophobic enzymes that transfer long-chain fatty acids to membrane-associated hydroxyl acceptors, called “MBOATs” for Membrane-Bound O-Acyltransferases (Hofmann 2000). Examples include acyl-CoA:cholesterol acyltransferases (ACATs), which attaches fatty acids to the hydroxyl group of cholesterol and diacylglycerol acyltransferases (DGATs), which acylate the hydroxyl group of diacylglycerol. Subsequent studies indeed showed that Porcupine is required for the attachment of a monounsaturated long-chain fatty acid to a serine residue in Wnt (Takada et al., 2006).
Here, we show that the mammalian genome encodes 16 MBOATs produced by 11 genes, and we show that one of these MBOATs catalyzes the octanoylation of ghrelin when it is expressed together with prepro-ghrelin in cultured mammalian endocrine cell lines. We name this enzyme GOAT (Ghrelin O-Acyltransferase).